Gellan gum is a bacterial exopolysaccharide of natural origin prepared by aerobic submerged fermentation of Sphingomonas elodea. Gellan gum is a linear and anionic polymer composed of repeating units of a tetrasaccharide (1,3-β-D-glucose, 1,4-β-D-glucuronic acid, 1,4-β-D-glucose, 1,4-α-L-rhamnose), similar to the glycosaminoglycans existing in the extracellular matrix.
Gellan gum hydrogels can be formed by two different ways: by temperature decrease lower critical solution, and by the addition of ions. Hence, gellan gum hydrogels are termed thermoreversible hydrogels as they respond to temperature decrease with a sol-gel transition. In fact, gellan gum has a thermally reversible coil form at high temperatures which upon temperature decrease, changes to double-helix that anti-parallel self-assemble in the form of oriented bundles. These, called junction zones that per se link untwined regions of extended helical chains, lead to the formation of a three dimensional network, the hydrogel. In addition, the use of counterions, specifically monovalent or divalent cations, promotes a physical bonding between cations and carboxylate groups of the gellan gum, particularly strong when involving divalent ions, leading to the formation of the three dimensional and reticulated hydrogel. Due to this dual formation mechanism, gellan gum hydrogels have great potential for biomedical applications because they can gellify in situ in vivo.
WO Patent 2009/101518 A2 from 20 Aug. 2009 discloses for the first time the use of gellan gum hydrogels for regenerative medicine and tissue engineering applications, the system, and processing devices. It refers the possibility of using cell/bioactive agents within hydrogels, by including these components while homogenizing the matrix along the gelation. Accordingly, gellan gum hydrogels are particularly attractive to tissue engineering because of their possibility to encapsulate cells/biochemical molecules. Similarly to what happens in most of cell niches of the majority of tissue engineering applications, cells in hydrogels should attach, form focal contacts and organize their own cytoskeleton to spread and to be able to proliferate. However, hydrogels, including gellan gum hydrogels, hardly present cell adhesion properties as they lack cell anchorage points and/or are highly hydrophilic promoting water molecules bounding to the polymer backbone, thus inhibiting cell adhesion (Chang and Wang 2011). Furthermore, most of the hydrogels are composed of negatively charged polymers, known to repulse negatively charged cells and to limit the adsorption of cell adhesive proteins. To overcome this cell adhesion limitation, different approaches have been proposed. One of the strategies used includes the incorporation, within the polymeric matrix, of extracellular matrix (ECM) molecules, such as collagen, thrombospondin, osteopontin, fibronectin and vitronectin, which are known to promote cell adhesion. Moreover, the improvement of the adsorption of fibronectin and vitronectin glycoproteins to the polymer backbone, as well as other proteic components of serum, routinely used for cell culture, has been another approach used to improve cell adhesion to hydrogels (von der Mark, Park et al. 2010; Chang and Wang 2011). Similarly, peptide sequences, namely RGD, IKVAV or YIGSR, present on those glycoproteins, have been incorporated into the backbone of polymers, by chemical modification prior to the formation of the hydrogels. In fact, US Patent 2007/A1 from 19 Jul. 2009 discloses the use of RGD peptide sequence to promote the adhesion of cells within hydrogels. The use of proteins from animal sources introduces immunogenicity and disease transmission concerns that from a clinical perspective and from the regulatory point of view might never be overcome. Additionally, polymer modifications with proteins and/or peptides sequences are not only time-consuming but also implicate significant costs associated with the use of recombinant bioactive molecules.
The gellan gum dried polymeric structures (xerogels) quickly gain spongy-like hydrogel properties after re-hydration, not observed in either hydrated xerogels (US Patent 2007/0031499 A1), hydrated lyophilized gellan gum hydrogels (U.S. Pat. No. 7,147,885 B2), or hydrated xerogel/film comprising cellulose ether and gellan gum (WO Patent 2006/037606 A2). These patents make no reference to the use of cells and/or bioactive molecules.
US Patent 2007/0031499 A1 from 8 Feb. 2009 discloses the use of xerogels and the incorporation of pharmaceuticals while preparing the hydrogels. No reference is made to the re-hydration of the xerogels, with the use of cells or/and bioactive agents. Authors refer the inclusion of pharmaceuticals in xerogels with techniques to maintain the dryness of the xerogel and not in its hydrated state. Due to the drying processing conditions that might be harsh, the properties of the drugs can be altered. In the present invention the drugs and/or bioactive molecules are incorporated in the dried polymeric networks (xerogels) after freeze-drying, at the time of re-hydration, avoiding chemical alterations affecting its activity. This patent makes no reference to the use of cells and/or bioactive molecules.
U.S. Pat. No. 7,147,885 B2 from 12 Dec. 2006 discloses the use of native gellan gum, based on its multifunctionality, like in food. The patent also refers that dehydrated gels and jellies can be prepared from gels of gellan gum, by hot air current or freeze-drying. However, when water is added to this dehydrated hydrogel (dehydrogel) and the mixture is allowed to stand, the dehydrogel readily absorbs a large amount of water and swells depicting physical properties not much different from the properties of the original hydrogel prior drying. In the present invention, the spongy-like hydrogels, the dried structure after the re-hydration, accomplish physical properties dissimilar from the previous precursor hydrogels. This patent makes no reference to the re-hydration of the lyophilized hydrogels with cells and/or bioactive molecules.
WO Patent 2006/037606 A2 from 13 Apr. 2006 discloses the use of a xerogel/film comprising cellulose ether and gellan gum that can be used as a dry storage form of the gel or as a hydrogel after hydration with an aqueous solution that can be loaded with active ingredients for controlled rate, or in contact with aqueous body fluids. Again, the hydrated xerogel, as described by the authors “swell and form hydrogels when in contact with aqueous solutions”, maintaining the same properties observed on the precursor hydrogels. Spongy-like hydrogels have different physical properties relatively to hydrogels, in terms of morphology, microstructure, and mechanical properties. Spongy-like hydrogels have higher porosity, pore size and thicker pore walls, lower water content, higher physical stability and flexibility that results in a cell-adhesive character. Moreover, WO Patent 2006/037606 A2 claims the use of the mixture of both cellulose ether and gellan gum that “synergistically combine their benefits and complement one another to prevent their disadvantages observed when used alone”. In the present, we attain the main physical properties of spongy-like hydrogels only by using gellan gum, although other polymers can also be used along with gellan gum. This patent makes no reference to the re-hydration of the lyophilized hydrogels with cells.
The manuscript entitled “In vitro properties of gellan gum sponge as the dental filling to maintain alveolar space” from Chang et al. 2012 describes a dried gellan gum sponge prepared by the subsequent steps of gellan gum dissolution, freeze-drying, post chemical crosslinking, and finally freeze-drying. Chang et al. 2012 work only potentiate the use of the sponges in its dried state. No reference is made to the re-hydration of the gellan gum sponges with cells and/or bioactive molecules.
The fast water uptake and mechanical stability of gellan gum spongy-like hydrogels observed after its re-hydration was only similarly observed in superporous hydrogels (US 2009/0291115 A1).
US Patent 2009/0291115 A1 from 26 Nov. 2006 discloses the method of superporous hydrogel formation, which requires the addition of foaming agents while being processed, and the possibility of incorporating viable cells while preparing the hydrogel. Nevertheless, the cell performance, such as cell adhesion, was not referred. Gellan gum spongy-like hydrogels do not need any additional polymer/agent and crosslinking agent/condition to create the final polymeric architecture that enables a fast water uptake, mechanical stability and improved cell seeding, viability, adhesion, proliferation and differentiation.
WO Patent 2009/101518 A2 from 20 Aug. 2009 discloses the use of gellan gum hydrogels for regenerative medicine and tissue engineering applications, the system, and processing devices. No reference is made to the rehydration with cells and/or bioactive molecules.